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. 2020 Feb 25;9:e49917. doi: 10.7554/eLife.49917

Figure 1. daf-2b encodes a truncated insulin receptor that is capable of dimerization.

(A) Genomic organization of the daf-2 locus. Exons shaded in blue encode the α subunit (extracellular domain) and those in green encode the β subunit (transmembrane and tyrosine kinase domains). The alternate cassette exon utilized in daf-2c (exon 11.5) is shown in pink. The daf-2b transcript is predicted to arise if splicing at the exon 11.5 5’SS is skipped leading to the addition of 46 bp of intronic sequence (shown in yellow) before an in-frame stop codon is reached. F1, F2, R1, R2 and R3 indicate the location of primers used in cDNA amplification. (B) Details of the daf-2 genomic locus from exon 11 to exon 12. The sequence of 5’ and 3’ splice sites (SS) are indicated. Dotted lines indicate splicing events for daf-2a (red) and daf-2c (blue). Green solid lines indicate splicing events for generation of the daf-2b transcript. (C) PCR amplification of full-length daf-2b cDNA using primers F1 and R1 (panel A). M - molecular weight markers, L – larval stage, YA – young adults, CON – daf-2b cDNA from plasmid template. (D) PCR amplification of a daf-2b cDNA fragment encompassing exon 11 and the predicted 3’ UTR using primers F2 and R2 (panel A). M - molecular weight markers, E – embryos, L – larval stage, YA – young adults. (E) Multiplex PCR of daf-2a, daf-2b and daf-2c from pooled cDNA (lanes marked C/A, B and C/A/B) and larval stages including dauer (D) using primers F2, R1 and R3 (panel A). (F) Schematic illustrating the possible formation of DAF-2A and DAF-2B homodimers and DAF-2A/DAF-2B heterodimers via formation of disulfide bonds at conserved cysteine residues. (G) Coimmunoprecipitation of epitope tagged DAF-2A and DAF-2B indicates the capacity to dimerize. Immunoprecipitates were subjected to SDS PAGE and blotted with anti-MYC (top panel) and anti-HA (second panel). Whole cell lysates (WCL) were blotted with anti-MYC and anti-HA (bottom two panels). Coimmunoprecipitation data are representative of 3 independent experiments.

Figure 1.

Figure 1—figure supplement 1. Schematic of daf-2b cDNA.

Figure 1—figure supplement 1.

Nucleotide sequence corresponding to EC004351 EST cDNA is indicated in red text and the exon 11.5 5’ splice site is highlighted in bold. Primer sequences for amplification of the cDNA fragment shown in Figure 1C and D are indicated. PAS indicates the variant polyA signal.
Figure 1—figure supplement 2. Ratio of daf-2a to daf-2b.

Figure 1—figure supplement 2.

Multiplex PCR was performed within the linear range of amplification and band intensities were measured using Image J. Each larval stage was examined in triplicate except dauer (duplicate) and data are presented as mean+ sd.